Fig. 5: Inhibition of hedgehog signal suppresses re-expression of KRAS and reactivation of ERK in KRASG12C inhibitor–treated lung cancer cells.
From: Hedgehog signalling is involved in acquired resistance to KRASG12C inhibitors in lung cancer cells
H358 cells were co-treated with 10 μM ARS-1620 and 10 μM Smo inhibitor sonidegib for the indicated times. A Transcriptome profiles were analyzed by RNA-seq as described in Fig. 1 legends. Gene set enrichment plot of KRAS signaling negatively enriched in cells co-treated with ARS-1620 and the Hedgehog signal inhibitor sonidegib for 48 h versus cells treated with ARS-1620 for 48 h. B–I The Smo inhibitor sonidegib suppressed ARS-1620-induced Hedgehog signaling in both H23 (B–E) and H358 cells (F–I). Time-dependent changes in IFT88 (B, F), ARL6 (C, G), and GLI-1 mRNA levels (D, H) were determined by qRT-PCR amplification. Fold change in expression level was calculated relative to the values at time 0 for each cell. The graphs are mean ± standard deviation of three independent experiments (one-way ANOVA, **P < 0.01; ***P < 0.001). Time-dependent changes in IFT88, ARL6 and GLI-1 shown in H23 (E) and H358 cells (I) were determined by western blot analysis. J–O The Smo inhibitor sonidegib suppresses ARS-1620-induced primary cilia formation in both H23 (J–L) and H358 cells (M–O). Representative confocal microscopy images of H23 (J) and H358 cells (M) stained for acetylated tubulin (Ac-Tu, red), Arl13B (green), and DAPI (blue). K, L, N, O Graphs depict the percentages of ciliated H23 (K) and H358 cells (N) and average length of cilia of H23 (L) and H358 cells (O) and are presented as the mean ± standard deviation (n = 150 pooled from three independent experiments). Student’s t-tests, *P < 0.05; **P < 0.01; ***P < 0.001. P–S The Smo inhibitor sonidegib suppresses ARS-1620–induced re-expression and reactivation of ERK signal in lung cancer cells. Time-dependent changes in KRAS mRNA levels in H23 (P) and H358 cells (R) were determined by qRT-PCR amplification. Fold change in expression level was relative to the values at time 0 for each cell. The graphs are mean ± standard deviation of three independent experiments (one-way ANOVA, **P < 0.01; ***P < 0.001). Time-dependent changes in ERK phosphorylation in H23 (Q) and H358 cells (S) were determined by western blot analysis (T, U). The Smo inhibitor sonidegib suppressed the generation of cells resistant to KRASG12C inhibitors. At the indicated times, H23 (T) and H358 cells (U) were stained with crystal violet and photographed. Three independent experiments were performed and representative images are shown.
